Identification Of MicroRNA-21Targeting Genes TSC1and SMAD7Related To Development Of Muscle In Pig

In recent years, microRNA (miRNA) has become a hot spot. Because of its function in the early development of the organism, cell proliferation, differentiation, apoptosis, organogenesis and occurrence with disease, miRNA has a revolution influence on pharmacy research in the field of basic biomedical at one time. MiRNA is small non-coding RNAs composed of18-22nucleotides and regulating a variety of biological processes in the organism. Combining with the3’ untranslated region of target gene(s) completely or incompletely, miRNA play a role in inhibiting or degenerating the messenger RNA. By so far, many studies demonstrated that miRNA had an irreplaceable role in proliferation, differentiation, apoptosis, and regeneration of the myocyte. As constructing miRNA cDNA libraries of porcine myocytes, our research group discovered that expression of miRNA-21varied at different developmental time points. After Solexa high-throughput sequencing, we speculated that miRNA-21might involve in regulating of skeletal muscle growth and development by targeting downstream genes. Previous researches on miRNA-21were focused closely on cell proliferation, differentiation, apoptosis and tumorigenesis, but its function on muscle development remains illegibility. So it is necessary to identify target genes of miRNA-21.Using online tools such as Target Scan and Pic Tar and DAVID database, target prediction and GENE ONTOLOGY analysis were undertaken. The results revealed that TSC1and SMAD7were candidate target genes of miRNA-21during muscle development. After primers designs for the two genes, porcine3’UTR of TSC1and SMAD7genes were cloned by PCR, as well as3’UTR of TSC1and SMAD7with no binding domain of miRNA-21. Then two target genes were cloned to dual-luciferase vectors and corresponding dual-luciferase mutant vectors respectively, and vectors were transfected into PIEC and PK15cell lines. Lastly, luciferase activity was measured in dual-luciferase assay system. The results showed that luciferase activity was significantly different between the experimental group and the control group (P<0.05).We concluded that TSC1and SMAD7were target genes of miRNA-21and were downregulated by miRNA-21in PIEC and PK15cell lines. By pathway analysis in KEGG database, we speculated that miRNA-21might influence myocyte proliferation and differentiation, by targeting TSCl and SMAD7, and through mTOR signaling pathway and TGF-β signaling pathway. In conclusion, our research provided the basis for further study of miRNA-21on regulating the mechanism of muscle development and the treatment of muscle-related diseases.